Circulating CD14+CD16- classical monocytes do not associate with a vulnerable plaque phenotype, and do not predict secondary events in severe atherosclerotic patients.

AIMS: Mouse studies have established distinct monocyte subtypes that participate in the process of atherosclerotic lesion formation. The pro-inflammatory Ly6Chigh monocyte subtype actively contributes to murine plaque progression and destabilization. Also in humans, different peripheral monocyte subtypes have been identified, of which the CD14+CD16- classical monocyte is suggested to display similar pro-atherosclerotic properties as the murine Ly6Chigh subtype. We aimed to investigate if circulating CD14+CD16- classical monocytes associate with characteristics of a vulnerable carotid atherosclerotic plaque and if they associate with the risk of secondary adverse manifestations of atherosclerotic disease. METHODS AND RESULTS: We enrolled 175 carotid endarterectomy patients of the Athero-Express biobank in our study. Just prior to surgical procedure, blood was collected and peripheral blood mononuclear cells were isolated. Characterization of monocyte subsets was performed by flow cytometry. Plaque characteristics were semi-quantitatively scored for the presence of fat, collagen, intraplaque hemorrhage and calcification. Vessel density, smooth muscle cells and macrophages were assessed quantitatively on a continuous scale. All features of a vulnerable plaque phenotype, including low amounts of collagen and smooth muscle cells, and increased fat content, vessel density, intraplaque hemorrhage and plaque macrophages were not significantly associated with differential levels of peripheral classical CD14+CD16- monocytes or other monocyte subsets. Using Cox regression models to evaluate the prognostic value of circulating monocyte subtypes, we found that total counts of peripheral monocytes, as well as CD14+CD16- classical and other monocyte subtypes were not associated with the risk of secondary cardiovascular events during 3 years follow-up. CONCLUSION: Circulating classical CD14+CD16- monocytes do not associate with specific vulnerable plaque characteristics. In addition, they do not predict secondary adverse manifestations. This suggests that in patients with established carotid artery disease, the circulating monocytes do not reflect plaque characteristics and have no value in identifying patients at risk for future cardiovascular events.

Complement 5a Receptor deficiency does not influence adverse cardiac remodeling after pressure-overload in mice.

Hypertension is one of the most common risk factors for the development heart failure in the general population. Inflammation plays a central role in this adverse remodeling and eventually to the development of heart failure. Circulating levels of Complement factor 5a (C5a) are increased in hypertensive patients and the C5a receptor is associated with the presence of cardiac fibrosis and inflammation in an experimental hypertension model. To test if C5aR is involved in adverse cardiac remodeling following pressure-overload, we induced transverse aortic constriction (TAC) in wildtype and C5a receptor deficient mice (C5aR-/-). Six weeks after TAC, C5aR-/- animals showed a similar degree of cardiac hypertrophy and decrease in cardiac function as wild type mice (End Systolic Volume; 50.30±5.32 µl vs. 55.81±8.16 µl). In addition, other features of adverse cardiac remodeling like cardiomyocyte cell size (WGA staining), fibrosis (picrosirius red staining) or collagen degradation (matrix metalloproteinase activity assay) did not differ either. In conclusion, full body C5aR deficiency does not affect adverse cardiac remodeling after pressure-overload. However, our finding are in contrast with C5a inhibition studies. Our observations do present the role of C5a-C5aR in adverse cardiac remodeling and heart failure as controversial at the least.

Leukocyte-Associated Immunoglobulin-like Receptor-1 is regulated in human myocardial infarction but its absence does not affect infarct size in mice.

Heart failure after myocardial infarction (MI) depends on infarct size and adverse left ventricular (LV) remodelling, both influenced by the inflammatory response. Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an inhibitory receptor of ITAM-dependent cell activation, present on almost all immune cells. We investigated regulation of LAIR-1 leukocyte expression after MI in patients and hypothesized that its absence in a mouse model of MI would increase infarct size and adverse remodelling. In patients, LAIR-1 expression was increased 3 days compared to 6 weeks after MI on circulating monocytes (24.8 ± 5.3 vs. 21.2 ± 5.1 MFI, p = 0.008) and neutrophils (12.9 ± 4.7 vs. 10.6 ± 3.1 MFI, p = 0.046). In WT and LAIR-1-/- mice, infarct size after ischemia-reperfusion injury was comparable (37.0 ± 14.5 in WT vs. 39.4 ± 12.2% of the area at risk in LAIR-1-/-, p = 0.63). Remodelling after permanent left coronary artery ligation did not differ between WT and LAIR-1-/- mice (end-diastolic volume 133.3 ± 19.3 vs. 132.1 ± 27.9 µL, p = 0.91 and end-systolic volume 112.1 ± 22.2 vs. 106.9 ± 33.5 µL, p = 0.68). Similarly, no differences were observed in inflammatory cell influx or fibrosis. In conclusion, LAIR-1 expression on monocytes and neutrophils is increased in the acute phase after MI in patients, but the absence of LAIR-1 in mice does not influence infarct size, inflammation, fibrosis or adverse cardiac remodelling.

Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles.

Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood.

High Levels of (Un)Switched Memory B Cells Are Associated With Better Outcome in Patients With Advanced Atherosclerotic Disease.

BACKGROUND: Atherosclerosis is an inflammatory lipid disorder and the main underlying pathology of acute ischemic events. Despite a vast amount of data from murine atherosclerosis models, evidence of B-cell involvement in human atherosclerotic disease is limited. We therefore investigated the association of circulating B-cell subtypes with the occurrence of secondary cardiovascular events in advanced atherosclerotic disease. METHODS AND RESULTS: This cohort study consists of 168 patients who were included in the Athero-Express biobank between 2009 and 2011. Before surgery, peripheral blood mononuclear cells were isolated and stored in liquid nitrogen. After gentle thawing of the peripheral blood mononuclear cells, different B-cell subtypes including naïve, (un)switched memory, and CD27+CD43+ B1-like B cells, were analyzed by flow cytometry. Univariable and multivariable Cox proportional hazard models were used to analyze associations between B-cell subtypes, circulating antibodies and secondary cardiovascular manifestations during the 3-year follow-up period. Mean age was 70.1±9.6 years, males represented 62.8% of the population, and 54 patients had secondary manifestations during follow-up. High numbers of unswitched memory cells were protective against secondary outcome (hazard ratio, 0.30 [95% CI, 0.13-0.69]; P<0.01). Similar results were obtained for the switched memory cells that also showed to be protective against secondary outcome (hazard ratio, 0.33 [95% CI, 0.14-0.77]; P=0.01). CONCLUSIONS: A high number of (un)switched memory B cells is associated with better outcome following carotid artery endarterectomy. These findings suggest a potential role for B-cell subsets in prediction and prevention of secondary cardiovascular events in patients with atherosclerosis.

Variants in ALOX5, ALOX5AP and LTA4H are not associated with atherosclerotic plaque phenotypes: the Athero-Express Genomics Study.

BACKGROUND: The eicosanoid genes ALOX5, ALOX5AP and LTA4H have been implicated in atherosclerosis. We assessed the impact of common variants in these genes on gene expression, circulating protein levels, and atherosclerotic plaque phenotypes. METHODS: We included patients from the Stockholm Atherosclerosis Gene Expression study (STAGE, N = 109), and the Athero-Express Biobank Study (AE, N = 1443). We tested 1453 single-nucleotide variants (SNVs) in ALOX5, ALOX5AP and LTA4H for association with gene expression in STAGE. We also tested these SNVs for association with seven histologically defined plaque phenotypes in the AE (which included calcification, collagen, cellular content, atheroma size, and intraplaque vessel density and hemorrhage). RESULTS: We replicate a known cis-eQTL (rs6538697, p = 1.96 × 10(-6)) for LTA4H expression in whole blood of patients from STAGE. We found no significant association for any of the SNVs tested with serum levels of ALOX5 or ALOX5AP (p > 5.79 × 10(-4)). For atherosclerotic plaque phenotypes the strongest associations were found for intraplaque vessel density and smooth muscle cells in the ALOX5AP locus (p > 1.67 × 10(-4)). CONCLUSIONS: We replicate a known eQTL for LTA4H expression in whole blood using STAGE data. We found no associations of variants in and around ALOX5, ALOX5AP and LTA4H with serum ALOX5 or ALOX5AP levels, or plaque phenotypes. On the supposition that these genes play a causal role in atherosclerosis, these results suggest that common variants in these loci play a limited role (if any) in influencing advanced atherosclerotic plaque morphology to the extent that it impacts atherosclerotic disease.

Extracellular vesicle-derived CD14 is independently associated with the extent of cardiovascular disease burden in patients with manifest vascular disease.

BACKGROUND: In patients with established cardiovascular disease, high levels of the extracellular vesicle (EV)-derived proteins cystatin C, CD14, and α2-antiplasmin predict recurrent cardiovascular events. We examined whether these proteins are associated with the extent of vascular disease. METHODS: In 1062 patients from the SMART (Secondary Manifestations in ARTerial disease) study, EVs were isolated from plasma at baseline. Cystatin C, CD14, and α2-antiplasmin were measured in these vesicles using a multiplex assay. The extent of vascular disease burden was determined by a sum score that incorporates history and current presence of clinically manifest coronary, cerebrovascular, peripheral arterial, and abdominal aneurysm disease, and parameters of atherosclerosis that were assessed during the SMART screening protocol (ankle-brachial index, common carotid intima-media thickness, carotid stenosis, and aorta diameter). The relation between EV protein levels and extent of vascular disease was evaluated using ordinal multivariable regression models. RESULTS: EV-derived CD14 was significantly associated with the number of affected vascular territories (OR 2.4, 95% CI 1.4-4.1) as represented by the sum score, independently of cardiovascular risk factors. Cystatin C and α2-antiplasmin EV levels did not show an independent association with vascular disease extent. When investigating parameters of the sum score separately, we did not observe a strong association between any of the EV-derived proteins and the markers of atherosclerosis. CONCLUSIONS: EV-derived CD14 levels are strongly correlated to the extent of vascular disease, but not specifically to markers that reflect atherosclerosis burden, in patients with manifest cardiovascular disease.

Leukotriene B4 levels in human atherosclerotic plaques and abdominal aortic aneurysms.

BACKGROUND: Leukotriene B4 (LTB4) has been associated with the initiation and progression of atherosclerosis and abdominal aortic aneurysm (AAA) formation. However, associations of LTB4 levels with tissue characteristics and adverse clinical outcome of advanced atherosclerosis and AAA are scarcely studied. We hypothesized that LTB4 levels are associated with a vulnerable plaque phenotype and adverse clinical outcome. Furthermore, that LTB4 levels are associated with inflammatory AAA and adverse clinical outcome. METHODS: Atherosclerotic plaques and AAA specimens were selected from two independent databases for LTB4 measurements. Plaques were isolated during carotid endarterectomy from asymptomatic (n = 58) or symptomatic (n = 317) patients, classified prior to surgery. LTB4 levels were measured without prior lipid extraction and levels were corrected for protein content. LTB4 levels were related to plaque phenotype, baseline patient characteristics and clinical outcome within three years following surgery. Seven non-diseased mammary artery specimens served as controls. AAA specimens were isolated during open repair, classified as elective (n = 189), symptomatic (n = 29) or ruptured (n = 23). LTB4 levels were measured similar to the plaque measurements and were related to tissue characteristics, baseline patient characteristics and clinical outcome. Twenty-six non-diseased aortic specimens served as controls. RESULTS: LTB4 levels corrected for protein content were not significantly associated with histological characteristics specific for vulnerable plaques or inflammatory AAA as well as clinical presentation. Moreover, it could not predict secondary manifestations independently investigated in both databases. However, LTB4 levels were significantly lower in controls compared to plaque (p = 0.025) or AAA (p = 0.017). CONCLUSIONS: LTB4 levels were not associated with a vulnerable plaque phenotype or inflammatory AAA or clinical presentation. This study does not provide supportive evidence for a role of LTB4 in atherosclerotic plaque destabilization or AAA expansion. However, these data should be interpreted with care, since LTB4 measurements were performed without prior lipid extractions.

Sex is associated with the presence of atherosclerotic plaque hemorrhage and modifies the relation between plaque hemorrhage and cardiovascular outcome.

BACKGROUND AND PURPOSE: Plaque hemorrhage (PH) may lead to accelerated progression of atherosclerotic disease. The presence of local PH in the carotid plaque predicts future cardiovascular events in any vascular territory. We investigated the prevalence of local PH and the predictive value of PH for the occurrence of cardiovascular events in men and women separately. METHODS: Atherosclerotic plaques from 1422 patients (969 men, 453 women) who underwent carotid endarterectomy were analyzed histologically for the presence of PH. Patients were monitored for 3 years for cardiovascular events (nonfatal stroke, nonfatal myocardial infarction, vascular death, and vascular intervention). RESULTS: Plaques from men showed a significantly higher prevalence of PH compared with women (67% versus 54%; P<0.001). In 1353 patients with available follow-up data, with a median duration of 2.9 years, 270 events had occurred in men (29%) and 94 in women (22%). Stratified by presence of PH, the event rate was 32% in men with PH versus 23% in men without PH, and 23% in women with PH versus 21% in women without PH. A multivariable Cox proportional hazards model found a significant interaction between sex and PH. PH was significantly associated with events in men (adjusted hazard ratio, 1.9; 95% CI, 1.2-2.8) but not in women (adjusted hazard ratio, 1.0; 95% CI, 0.6-1.7). CONCLUSIONS: Atherosclerotic carotid plaques obtained from men reveal a higher prevalence of PH compared with women. Local PH is strongly related to secondary manifestations of cardiovascular disease in men but not in women.

Serum extracellular vesicle protein levels are associated with acute coronary syndrome.

AIMS: Biomarkers are essential in the early detection of acute coronary syndromes (ACS). Serum extracellular vesicles are small vesicles in the plasma containing protein and RNA and have been shown to be involved in ACS-related processes like apoptosis and coagulation. Therefore, we hypothesized that serum extracellular vesicle protein levels are associated with ACS. METHODS AND RESULTS: Three serum extracellular vesicle proteins potentially associated with ACS were identified with differential Q-proteomics and were evaluated in 471 frozen serum samples of ACS-suspected patients presenting to the emergency department (30% of whom had an ACS). Protein levels were measured after vesicle isolation using ExoQuick. Mean serum extracellular vesicle concentration of the different proteins was compared between ACS and non-ACS patients. Selected proteins were tested in a univariate logistic regression model, as well as in a multivariate model to adjust for cardiovascular risk factors. A separate analysis was performed in men and women. In the multivariate logistic regression analysis, polygenic immunoglobulin receptor, (pIgR; OR 1.630, p=0.026), cystatin C (OR 1.641, p=0.021), and complement factor C5a (C5a, OR 1.495, p=0.025) were significantly associated with ACS, while total vesicle protein concentration was borderline significant. The association of the individual proteins with ACS was markedly stronger in men. CONCLUSIONS: These data show that serum extracellular vesicle pIgR, cystatin C, and C5a concentrations are independently associated with ACS and that there are pronounced gender differences. These observations should be validated in a large, prospective study to assess the potential role of vesicle content in the evaluation of patients suspected of having an ACS.

Mast cells in human carotid atherosclerotic plaques are associated with intraplaque microvessel density and the occurrence of future cardiovascular events.

AIMS: Human autopsy, animal, and cell culture studies together have merged in a concept suggesting participation of mast cells (MCs) in the generation of atherosclerotic plaques. More specifically, these studies have suggested MC-induced intraplaque neovascularization as one mechanism by which MCs may render the plaques vulnerable. The present study was designed to assess the association between MC numbers and neovascularization in human atherosclerotic plaques, and to relate the abundance of plaque MCs to the occurrence of adverse cardiovascular events during the follow-up. METHODS AND RESULTS: Atherosclerotic plaques of 270 patients suffering from carotid artery stenosis were stained for the presence of MCs (MC tryptase). Furthermore, during a follow-up of 3 years, cardiovascular-related endpoints were assessed in 253 patients. On average a high number of MCs were observed per plaque cross-section [median 108 (55-233) cells per section]. Plaques with high MC numbers revealed an unstable lipid-rich inflammatory phenotype and were associated with symptomatic patients. In addition, MC numbers were positively associated with microvessel density (r = 0.416, P < 0.001). Patients with high intraplaque MC numbers showed significantly more cardiovascular events during the follow-up (58/142 vs. 31/111 events, P = 0.029). In a multivariate analysis with correction for the main risk factors of cardiovascular diseases, MCs remained independently associated with adverse cardiovascular events (P = 0.025). CONCLUSION: Mast cells are highly prevalent in human carotid atherosclerotic lesions and associated with plaque microvessel density. Furthermore, intraplaque MC numbers associate with future cardiovascular events.